Use of nitroxides for the treatment of essential hypertension

ABSTRACT

The present invention encompasses methods of treating patients for essential hypertension. The invention also includes related pharmaceutical compositions of nitroxides. Specific drugs, such as 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol) are disclosed. These compositions are also contemplated for use in the treatment of oxidative stress and modulation of blood pressure.

This is a 371 of PCT/US/98/19586 filed Sep. 21, 1998 which is acontinuation of 08/933,379 filed Sep. 19, 1997 now U.S. Pat. No.6,096,759.

The work leading to this invention was partially funded by the UnitedStates Government under NIH grants DK 36079 and DK 49870.

FIELD OF THE INVENTION

This invention relates to the treatment of essential hypertension byadministration of anti-hypertensive effective amounts of nitroxides,such as 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol or TMPN).

BACKGROUND OF THE INVENTION

Systemic hypertension is the most prevalent cardiovascular disorder inthe United States, affecting more than 50 million individuals.Accordingly, efforts to prevent, diagnose and treat hypertension remainan important concern of national health care. Although major advanceshave been made as to public awareness of the importance of hypertension,introducing antihypertensive therapies and in controlling hypertension,the adverse metabolic effects of some classes of antihypertensive drugsand the disappointing results in preventing associated coronary diseasehave challenged the traditional approaches of treating theantihypertensive patient.

Essential Hypertension

Essential hypertension represents a collection of genetically baseddiseases and/or syndromes with a number of underlying inheritedbiochemical abnormalities which have yet to be elucidated. Hypertensionleads to atherosclerosis and other forms of vascular pathology bydamaging the endothelium. Endothelial damage produces a cascade ofchanges. Additionally, non-atherosclerotic hypertension-induced vasculardamage can lead to stroke and end-stage renal disease. The measurementof blood pressure (BP) is one of the major methods of diagnosingessential hypertension. However, blood pressure is just one factor thatis taken into account in the choice of therapy and drug selection forthe treatment of the complex pathology which is essential hypertension.

Reactive Oxygen Species

Reactive oxygen species (ROS), such as superoxide anions (O₂ ⁻),hydroxyl radicals, and hydrogen peroxide (H₂O₂) have been implicated inatherosclerosis, diabetes, ischemia-reperfusion injury and hypertension(Giugliano et al., 1995 Metab. 44: 363-368; Harrison et al., 1995 Am. J.Cardiol. 75: 75B-81B; McCord et al., 1985 312: 159-163; and Kitiyakaraet dl., Curr. Opin. Nephrol Hypertens. In press). Compared tonormotensive individuals, hypertensive patients have higher plasmahydrogen peroxide, superoxide anion and lipid peroxides while havinglower levels of the antioxidant, ascorbic acid (Lacy et al., 1998 J.Hypertens. 16: 291-303; Kumar et al., 1993 Free Rad. Res. Commun. 19:59-66; Tse et al., 1994 J. Hum. Hypertens. 89: 843-849; Bulpitt et al.,J. Hypertens. 8: 1071-1075). However, the molecular mechanism for oxygentoxicity in vascular diseases, such as essential hypertension, remainsto be elucidated.

Enzymes such as superoxide disumutase (SOD) catalyze the dismutation ofsuperoxide radicals to remove the radicals from the subject's systemwhere the radicals can destroy tissue. Experiments in which SOD wasadministered to rats did not reduce blood pressure in either normal ratsor in spontaneously hypersensitive rats (SHR). However, an SOD fusionprotein and oxypurinol, an inhibitor of xanthine oxidase, whenadministered separately have decreased blood pressure in SHR, but notnormal rats (Nakazono et al., 1991 Proc. Nat'l Acad. Sci. USA 88:10045-10048). Other short term studies of ROS inhibition have indicatedthat blood pressure can be reduced in the SHR animal model (Yoshioka etal., 1985 Int. J. Vitam. Nutri. Res. 55: 301-307; Nakazono et al., 1991Proc. Natl Acad. Sci. USA 88: 10045-10048; Suzuki et al., 1998 Proc.Natl Acad. Sci. USA 95: 47544759; Susuki et al., 1995 Hypertens. 25:1083-1089). Another antioxidant, ascorbic acid, has also lowered theblood pressure level in the SHR animal model. However, the SHR model hasalso indicated abnormalities in ascorbic acid metabolism (Yoshioka etal., 1985 Internat. J. Vit. Nutr. Res. 55: 301-307). This evidenceindicates that superoxide radicals in and around vascular endothelialcells play roles in the pathogenesis of hypertension in the SHR model.

Nitric oxide (NO), also known as endothelium-derived relaxing factor(EDRF), is synthesized by nitric oxide synthase (NOS) in many types ofcells including vascular endothelial cells, vascular smooth musclecells, activated macrophages, neuronal cells and glial cells (Miyamotoet al., 1996 Proc. Soc. Exp. Biol. Med. 211: 366-373). Gryglewski etal., (1986 Nature 320: 454-456) showed that O₂ ⁻ reacts with NO to formthe potentially toxic molecular species, peroxynitrite (ONOO⁻), whichcan effectively deplete NO in vascular endothelial cells. Rubanyi etal., (1986) demonstrated that O₂ ⁻ inactivates EDRF in coronary arteryrings (Am. J. Physiol. 250: H822-H827.) Scavenging of O₂ ⁻ enhancesendothelium-dependent vasodilation and increases NO release frommesenteri arterioles (Tschudi et al., 1996 Hypertens. 27: 32-35) andendothelial cells (Grunfeld et al., 1995 Hypertens. 26: 854-857) in SHR.However, the complete mechanism for the vasodilatory actions of O₂ ⁻scavengers has yet to be elucidated. Specifically, although in vitroevidence exists suggesting that O₂ ⁻ contributes to increased systemicvascular tone in the SHR, the role of O₂ ⁻ in increased renal vascularresistance (RVR) and baseline mean arterial pressure (MAP) of SHR invivo remains unclear (Schnackenberg et al., 1998 Hypertens. 32: 59-64).

There are many other examples in which severe oxidative stress is foundwithout hypertension. These include, for examples, poisoning with carbontetrachloride, diabetes mellitus and hypercholesteremia. Indeed, theevidence for oxidative stress in these conditions is better than inhypertension. Therefore, the finding that correction of oxidative stressalso reduced blood pressure is not predictable (Kitiyakara et al., inpress).

Nitroxides

Nitroxides have been used in ameliorating the deleterious effects oftoxic oxygen-related species such as O₂ ⁻ (see Mitchell et al., 1995U.S. Pat. No. 5,462,946; Hsia 1997 and 1998 U.S. Pat. Nos., 5,591,710;5,725,839; 5,741,893; 5,767,089; 5,804,561; and 5,807,831; and Lee etal., 1996 U.S. Pat. No. 5,516,881). Some nitroxides, such as Tempol(4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy), have been indicatedfor use in treating renal hypertension disorders (Carney et al., 1997U.S. Pat. No. 5,622,994; WO 92/22290). However, renal hypertensionoccurs as a result of reduced blood flow to the kidney and is notessential hypertension.

SUMMARY OF THE INVENTION

The treatment of essential hypertension has long presented a seriousproblem to the medical profession. This invention proposes a new methodof treating essential hypertension in a subject, such as a human, usingcompositions containing nitroxides. The nitroxides contemplated for usein the treatment of essential hypertension include nitroxides selectedfrom the group consisting of TEMPO, DOXYL or PROXYL nitroxides. Onepreferred nitroxide is 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy(Tempol).

This invention also contemplates a method of treating a patient withessential hypertension comprising the step of administering a bloodpressure lowering amount of a nitroxide, preferably in admixture with apharmaceutically acceptable carrier and/or an excipient. This methodutilizes the same nitroxides and methods of administration as describedabove.

Methods of administering pharmaceutical compositions containingnitroxides for treatment of essential hypertension include oral,transdermal, parenteral and intravenous routes of administration.

This invention also provides for a method of treating essentialhypertension comprising the steps of administering to a subject, such asa human, any of the above pharmaceutical compositions in combinationwith a second anti-hypertensive agent (e.g., benzothiadiazine diuretics,loop diuretics, potassium-sparing diuretics, sympatholytic agents,angiotensin-converting enzyme inhibitors, calcium channel blockingagents, direct vasodilators, as well as other antioxidants).

The invention likewise provides ranges of disclosed agents such asTempol to be administered to a subject for the treatment of essentialhypertension. The pharmaceutical compositions of Tempol to beadministered to a subject include an intravenous dose of from about 0.07mg/kg/hr to about 750 mg/kg/hr; an intravenous bolus dose of from about0.025 mg/kg/day to about 400 mg/kg/day; and an oral dose from about 0.05mg/kg/day to about 1,000 mg/kg/day.

This invention also contemplates simultaneously treating essentialhypertensive and oxidative stress comprising the step of administering apharmaceutical composition comprising an effective amount of anitroxide, such as Tempol. Such composition could further compriseadditional antihypertensives and/or No providing agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. MAP during baseline conditions (Basal) and during bolusinjection of Tempol (24 and 72 μmol/kg i.v.) in anesthetized WKY (□, =6)and SHR (, n=6). * P<0.05 vs. Basal; †P<0.05 vs. WKY.

FIG. 2. MAP during baseline conditions (Basal) and during intravenous(i.v.) infusion of Tempol (1.8, 18, 180 and 1,800 μmol·kg⁻¹·h⁻¹) inanesthetized WKY (□, =6) and SHR (, n=6). *P<0.05 vs. Basal; †P<0.05vs. WKY.

FIG. 3. Percent change in MAP after 7 days of Tempol administration (1.5mmol·kg⁻¹·h⁻¹ i.p.) in WKY (n=7) and SHR (n=7). †P<0.05 vs. WKY.

FIG. 4. Mean arterial pressure (MAP) in WKY and SHR during controlconditions (Control) and after two weeks of oral Tempol treatment (1 mM,Tempol, added to the drinking water). *p<0.05 vs. WKY. #p<0.05 vs.Control.

DESCRIPTION OF THE INVENTION

Providing novel effective methods of treating essential hypertension isgreatly needed by the millions who suffer from the disease. Thisinvention provides a method of controlling essential hypertension byadministration of a nitroxide by itself in combination with anotherantihypertensive agent or antioxidants.

1. Definitions

By “essential hypertension” is meant essential primary or idiopathichypertension which is a systemic hypertension of an unknown cause.Essential hypertension is the cause of 95% of all cases of hypertensiondiagnosed. It includes hypertension of all grades, including borderline,mild moderate and severe. It also includes hypertensive urgencies andemergencies or hypertensive crises, and indeed all cases of hypertensionwhere there is not a known cause. Secondary hypertension is systemichypertension of a known and reversible cause. Secondary causes arelargely those due to renal or renal artery diseases or endocrinedisorders. These account for fewer than 2-10% of the diagnosed cases ofhypertension.

By “blood pressure lowering amount” is meant that amount of a compoundthat produces a therapeutically effective concentration significantlydecreasing the blood pressure of a subject. A clinically significantreduction of BP would be a fall in BP of greater than 5% relative toeither the patient's normal base line BP or the patient's BPs underplacebo therapy.

By “effective concentration of Tempol” or “effective concentration of anitroxide” is meant that concentration of Tempol or nitroxide whichsignificantly lowers the blood pressure of the hypertensive subject. Ina human subject, normal diastolic blood pressure (BP) is considered tobe normal when on two visits to the physician the diastolic BP is below90 mm Hg or the systolic blood pressure on two visits to the physicianis below 140 mm Hg. In certain circumstances, such as patients withheavy proteinuria or diabetic nephropathy, a lower goal for BP of120-125 (systolic) and 70-75 (diastolic) mm Hg is considered optimalcurrently. The definitions, goals of therapy, and uses of conventionalanti-hypertensive agents have recently been summarized in a consensusdocument published by the National Institutes of Health entitled theSixth Report of the Joint Commission on the Detection, Evaluation andTreatment of Hypertension (National Institutes of Health, NationalHeart, Lung and Blood Institute, 1998).

The “nitroxide compounds,” which may be useful in the present invention,will be structurally diverse because the requisite property of thenitroxides is their ability to mimic superoxide dismutase (SOD) andcatalase activity via the nitroxide free radical. The main requirementof the nitroxide compound is the presence of a stable free radical.Therefore, the nitroxides described in this invention include stablenitroxide free radicals, their precursors, and their derivatives in aheterocyclic or linear structure, as represented by the general formula:

where R₁ and R₂ combine together with the nitrogen to form aheterocyclic group; and wherein the atoms in the heterocyclic group maybe all carbon atoms, or may be carbon atoms as well as one or more N, O,and/or S atoms (such as, but not limited to a pyrrole, imidazole,oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine,pyrimidine, or purine, or derivatives thereof). The heterocyclic groupis preferably a 5-membered ring (such as PROXYL, or pyrroline) or a6-membered ring (such as piperidinyl or TEMPO), with substitution at thecarbon alpha to the nitrogen by electron-donating groups, which mayinclude straight or branched chain alkyl or aryl groups, preferablymethyl or ethyl groups, although other longer carbon chain species couldbe used.

In a more preferred embodiment, the TEMPO, DOXYL or PROXYL nitroxides ortheir derivatives may be used, as shown below:

The TEMPO, DOXYL or PROXYL nitroxides may or may not be substituted atany atom, other than the nitrogen bearing the oxygen free radical, withany combination of at least one of the following substituents:acetamido, aminomethyl, benzoyl, 2-bromoacetamido,2-(2-(2-bromoacetamido)ethoxy)ethylcarbamoyl, carbamoyl, carboxy, cyano,5-(dimethylamino)-1-naphthalenesulfonamido, ethoxyfluorophosphinyloxy,ethyl, 5-fluoro-2, 4-dinitroanilino, hydroxy, 2-iodoacetamido,isothiocyanato, isothiocyanatomethyl, methyl, maleimido, maleimidoethyl,2-(2-maleimidoethoxy)ethylcarbamoyl, maleimidomethyl, maleimido, oxo,and phosphonooxy. The TEMPO, DOXYL or PROXYL nitroxides may also besubstituents on, for example, 17β-hydroxy-5α-androstane, decane,nonadecane, 5α-cholestane, stearic acid. In the alternative, the TEMPO,DOXYL or PROXYL nitroxides may form the methyl, ethyl, or propyl esterwith stearic acid. Additional nitroxides that are within the scope ofthe present invention are discussed in U.S. Pat. Nos. 5,462,946 and5,591,710, which are herein incorporated by reference.

The most preferred embodiment of the invention for the treatment ofessential hypertension are the nitroxides,4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol) or lesspreferred, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO).

The compounds of the invention include nitroxide compounds that can beadministered via either the oral, parenteral or topical routes and otherroutes of administration known to those skilled in the art. In general,these compounds are most desirably administered in the dosages discussedin Example 2, although variations will necessarily occur depending uponthe weight, age, and condition of the subject being treated and thepresence of co-morbid conditions that may affect the pharmokokinetics orpharmokodynamics of the agents. These will vary according to theparticular route of administration chosen. Other variations may alsooccur depending upon the species of animal being treated and itsindividual response to said medicament, as well as on the type ofpharmaceutical formulation chosen, and the time period and interval atwhich such administration is carried out. In some instances, dosagelevels below the lower limit of the aforesaid range may be more thanadequate, while in other cases still larger doses may be employedwithout causing any harmful side effects, provided that such largerdoses are first divided into several small doses for administrationthroughout the day or via sustained release formulations, or bycontinuous administration by intravenous infusion or dermal application.For example, tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over alonger period. Potential time delayed materials include glycerylmonostearate or glyceryl distearate. They may also be coated by thetechniques described in U.S. Pat. Nos. 4,256,108; 4,166,452; and4,265,874 to form osmotic therapeutic tablets for control release.

The compounds of the invention may be administered alone or incombination with pharmaceutically acceptable carriers of diluents by anyof the routes previously indicated, and such administration may becarried out in single or multiple doses. More particularly, the noveltherapeutic agents of this invention can be administered in a widevariety of different dosage forms, i.e., they may be combined withvarious pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like. Such carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Moreover,oral pharmaceutical compositions can be suitably sweetened and/orflavored.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (e.g., preferably corn, potato or tapioca starch),alginic acid and certain complex silicates, together with granulationbinders such as polyvinylpyrrolidone, sucrose, gelatin, and acacia.Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate and talc are often very useful for tableting purposes.Solid compositions of a similar type may also be employed as fillers ingelatin capsules; preferred materials in this connection also includelactose or milk sugar, as well as high molecular weight polyethyleneglycols.

When aqueous suspensions and/or elixirs are desired for oraladministration, the active ingredient may be combined with varioussweetening or flavoring agents, coloring matters or dyes, and, if sodesired, emulsifying and/or suspending agents as well, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouslike combinations thereof. Aqueous suspensions may also contain theactive materials in admixture with excipients suitable for aqueoussuspensions. Useful suspending agents include, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example, lecithin or condensation products of analkylene oxide with fatty acids (e.g., polyoxyethylene stearate), orcondensation products of ethylene oxide with long chain aliphaticalcohols (e.g. heptadecaethyleneoxycetanol), or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol (e.g., polyoxyethylene sorbitol monooleate), or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspensions may also contain one or more preservatives (e.g.,ethyl or n-propyl p-hydroxybenzoate).

For parenteral administration, solutions of a therapeutic compound ofthe present invention could be formulated as a ready to use solution inan isotonic vehicle of normal saline containing suitable stabilizers.The active agent may also be formulated as a dry, sterile powder or as alyophilized powder which would require reconstitution with an acceptableisotonic, sterile liquid. These aqueous solutions are suitable forintravenous, intramuscular, or subcutaneous injection purposes. Thepreparation of all these solutions under sterile conditions is readilyaccomplished by standard pharmaceutical techniques well known to thoseskilled in the art. These preparation can also be used in combinationwith other antihypertensive agents such as diuretics.

Any of the above methods of administering the active ingredients (e.g.nitroxides) is contemplated to treat subjects suffering from essentialhypertension (also known as primary or idiopathic hypertension). It iscontemplated to use these agents in patients with all grades ofhypertension from borderline to severe, in patients with accelerated ormalignant hypertension, and in those patients with hypertensiveurgencies, emergencies or crises. In treating higher grades ofhypertension or those categories outlined above, the combination ofanti-hypertensive and free radical scavenging properties may beespecially beneficial. Intracellular- and extracellular-oxidative stressis hypothesized to be a critical link between hypertension and theatherosclerotic complications of vascular disease that lead tomyocardial infarction, stroke and peripheral vascular disease. Thus,therapy directed simultaneously at hypertensive and intracellular andextracellular oxidative stress mediated by oxygen radicals (O₂ ⁻) andother reactive oxygen species (ROS) is highly attractive. The compoundsand formulations containing these compounds are also considered for useto lower blood pressure and to reduce oxidative stress. These compoundsshould be utilized in combination with lifestyle modifications (e.g.,weight reduction, alcohol restriction, exercise, restricting dietarysodium intake, supplementing dietary calcium and potassium and magnesiumintake, special diets, caffeine restriction and smoking cessation) andother strategies to combat oxidative stress (for example, vitamin E andC, selenium, chromium) and general measures to improve oxygenhomeostasis so as to limit ongoing oxygen radical production (e.g.,vasodilator therapy, appropriate use of nitric oxide and NO donors) tofurther prevent complications arising from hypertension and oxidativestress.

The pharmaceutically acceptable composition comprising nitroxides canfurther be combined with other anti-hypertensive agents.Antihypertensive agents include benzothiadiazine diuretics (e.g.,thiazides, phthalimidines and quinazolines), loop diuretics (e.g.,furosemide, ethacrynic acid and bumetanide), potassium-sparing diuretics(e.g., spironolactone, triamterene and amiloride), sympatholytic agents(e.g., centrally acting agents such as methyldopa; β-adrenergic blockingagents such as propranolol; α-adrenergic blocking agents such asprazosin; mixed α- and β-adrenergic blocking agents such as labetalol;ganglion blocking agents such as mecamylamine; and peripherally actingsympatholytic agents such as guanethidine), angiotensin-convertingenzyme inhibitors (e.g., captopril, enalapril, lisinopril, quinapril,ramipril, benazepril, fosinopril, spiropril, perindopril, andmoexipril), calcium channel blockers (e.g., nifedipine, etc.), anddirect vasodilators (e.g., sodium nitroprusside, hydralazine andminoxidil). For additional active agents to be used in combination withnitroxides, see CECIL TEXTBOOK OF MEDICINE 264-265 (20th ed., J. C.Bennett and F. Plum editors, W. B. Saunders Co., Philadelphia 1996).

Other pharmaceutical compositions include combinations of a nitroxideand an NO providing reagent (e.g., NO generating agents and NO donors).Preferred NO providing agents include: sodium nitroprusside (Nipride),S-nitrosoacetylpenacillamine (SNAP),3-morpholino-synonimin-hydrochloride (SIN-1),3-morpholino-N-athoxycarbonyl-sydnonimin (molsidomin), amyl nitrite(isoamyl nitrite), nitroglycerin (glyceryl trinitrite), isosorbidedinitrate (Isodil), isosorbide-5-mononitrite (Imur), and erythrityltetranitrate (cardilate). Other agents which are NO generating or are NOdonors could also be utilized in combination with nitroxides such asTempol to treat essential hypertension and oxidative stress.

To evaluate the recent finding that an absent TGF response to NOSblockade in the salt-restricted Sprague-Dawley rat could be restored bylocal microperfusion of L-arginine into the JGA, several studies wereperformed. As a part of the study, defective NO action in the JGA of SHRwas assessed from the TGF response to microperfusion of the lowmolecular weight nitroxide,4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol). Tempol is anonmetal, cell membrane-permeable superoxide dismutase (SOD) mimeticthat can protect against cardiac reperfusion damage or cardiomyocyteoxidative damage (Iannone et al, 1989 Biochem. Pharm. 38: 2581-2586;Nilsson et al., 1989 J. Biol. Chem. 19: 11131-11135). Tempol has beendemonstrated to be a stable spin trap for O₂ ⁻ and to reduce O₂ ⁻related injury resulting from ischemia/reperfusion, inflammation andradiation (Goffinan et al., 1992 Radiat. Oncol. Biol. Phys. 22: 803-806;Hahn et al, 1992 Cancer Res. 52: 1750-1753; and Mitchell et al., 1991Arch. Biochem. Biophys. 289: 62-70).

The following working examples which disclose methods of administeringnitroxide compositions therefore, specifically point out preferredembodiments of the present invention. These examples are not to beconstrued as limiting in any way the scope of the invention. Otherexamples using other formulations containing nitroxides alone or incombination with one or more antihypertensive agents and/or antioxidantswill be apparent to one skilled in the art. Assays analogous to thosedescribed below can be utilized in examining the efficacy of theseformulations for the treatment of essential hypertension.

EXAMPLE 1

Materials and Methods. Studies were undertaken on male SHR and WKY,weighing 235-300 g, and maintained on a standard rat chow (Purina RatChow, St. Louis, Mo.) with a sodium content of 0.3 g·100 g⁻¹. They wereallowed free access to food and water until the day of study.

Series 1: RT-PCR analysis of mRNA abundance of ecNOS and bNOStranscripts in glomeruli or renal cortex of SHR and WKY. As the amountof NOS may play a role in hypertension, especially in the SHR model,these studies were designed to test the hypothesis that transcripts forconstitutive NOS were diminished in the cortex of the SHR as compared toWKY rats. bNOS transcripts and protein are expressed abundantly in themacula densa of the renal cortex, and previous studies have shown aclose correlation between bNOS mRNA transcript abundance in renal cortexand bNOS transcript abundance in isolated macula densas. Accordingly,studies of bNOS mRNA expression were undertaken in outer cortical tissuewith the assumption that differences likely reflect changespredominantly in macula densa bNOS mRNA. Endothelial cell NOS (ecNOS)mRNA is more widely expressed in the vasculature, and therefore itsabundance was assessed in individual glomeruli that were microdissectedfrom outer cortical nephrons.

Under thiobarbital anesthesia (pentobarbital 100 mg·kg⁻¹ i.p.), theabdomen was opened and the aorta cannulated to allow flushing of thekidneys with ice-cold dissection solution. This fluid contained 135 mMNaCl, 1 mM Na₂HPO₄, at pH 7.4. For isolation of outer cortical kidneyRNA, one kidney from 6 SHR and one from 6 WKY was cut longitudinally,and a segment of outer cortex removed and digested with collagenase (1%)for 30 min at 37° C. Glomeruli were dissected under a stereomicroscopein rinse solution at 4° C. This contained (200 μl volume): 170 μldissection solution, 20 μl of 5 μM DTT, 10 μl of 10 mM vanadylribonucleoside complex. Dissected glomeruli were further cleaned inbuffer under stereomicroscope at 4° C. This contained (200 μl volume):170 μl dissection solution, 20 μl of 5 μM of DTT, and 10 mM of 2 U/μlRNA sin+. Finally, glomeruli were transferred to centrifuge tubescontaining lysis solution. The lysis solution contained (200 μl volume):166 μl deionized water, 4 μl of 2% Triton X-100, 20 μl of 5 mM DTT, and10 μl of 2 U/μl RNA sin+. Total RNA was extracted using RNA ATAT-60™(Tel-test B, Inc., Friendswood, Tex.). The mRNA was reverse transcribed(RT) with Oligo (dT)₁₆ as primer and MuLV reverse transcriptase using anRNA PCR Kit (Perkin Elmer, Inc., Branchburg, N.J.).

The primers used for PCR of the bNOS gene product were those describedpreviously. For bNOS, the sense primer was:5′-GTCGAATTCCGAATACCAGCCTGATCCATGGAA-3′(Seq. #1), and the antisenseprimer was 5′-CGCGGATCCCATGCGGTGGACTCCCTCCTGGA-3′(Seq. #2). Thepredicted product had a length of 599 base pairs. β-actin was selectedas a “housekeeper gene” for comparison. The primers used for β-actinmRNA were: sense primer 5′-GATCAAGATCATTGCTCCTC-3′(Seq. #3) andantisense primer: 5′-TGTACAATCAAAGTCCTCAG-3′(Seq. #4). The PCR producthad a predicted length of 426 bp. The amounts of NOS cDNAs werenormalized by the amounts of β-actin cDNA. The reaction mixturecontained 50 pmol of each primer, 1.25 mM deoxynucleotide mixture, 2.5μl Taq DNA polymerase, 10 mM Tris-HCl (pH 10), 50 mM KCl, 1.5 MM MgCl₂,0.001% (w/v) gelatin in a final volume of 50 μl. PCR was carried out asfollows: after an initial melting temperature of 94° C. for 4 min, therewas 30 sec of denaturation at 94° C.; 45 sec of annealing at 60° C.; and45 sec of extension at 72° C. for repeated cycles of amplification,followed by a final extension at 72° C. for 7 min. The PCR products wereanalyzed on a 1.5% agarose gel stained with ethidium bromide andvisualized under UV light. The size of the products were compared to arat kidney cDNA probe for bNOS. To verify the authenticity of the PCRproducts, the amplified bNOS cDNAs from the rat kidney cortex of an SHRand WKY rat were purified by MICROCON™ (Amicon Co., Beverly, Mass.) andsequenced with an AmliTaq cycle sequencing kit (Perkin Elmer, Inc.,Branchburg, N.J.).

Transcript abundance for ecNOS was assessed in single outer corticalglomeruli, isolated using the method of Pelayo et al. (1994) Am. J.Physiol. 267: F497-F503. Separate groups of SHR (n=6) and WKY (n=6) wereprepared as described above. For these studies, mRNA abundance wasexamined per single glomerulus. After anesthesia and preparation of theanimal, blue 1-5 μm latex microspheres (Polysciences, Warrington, Pa.)were infused in HEPES buffer (pH 7.4) into the left kidney. Afterperfusion, the kidney was excised, cut into coronal slices, placed onice, and a glomerulus from the outer cortex microdissected understereomicroscopy. Thereafter, the mRNA was extracted, reversetranscribed, and amplified as described above. The primers used forecNOS were: sense primer 5′-GTCGAATTCCTGGCGGCGGAAGAGAAGGAGC-3′(Seq. #5)and antisense: 5′-CGCGGATCCGGGGCTGGGTGGGGAGGTGATGTC-3′(Seq. #6). Thepredicted product had a length of 691 base pairs and was compared to arat kidney cDNA probe for ecNOS from our laboratory.

Care was taken to optimize conditions for the RT-PCR. For each study,parallel analyses were undertaken of serially diluted amounts of cDNA toensure that product (as assessed by densitometry) increased log-linearlywith cDNA amount in the ranges used. Negative controls were undertakenby PCR without prior RT, and by RT-PCR of the buffer used.

Series 2: Comparison of ecNOS, bNOS, and iNOS protein expression inkidneys of SHR and WKY. These studies in WKY and SHR rats wereundertaken to assess the hypothesis that changes in renal cortical genetranscript abundance were accompanied by changes in NOS gene translationproducts. Six SHR and six WKY rats were anesthetized, and their kidneysprepared as described above. Slices of kidney outer cortex weredissected and homogenized on ice in 1 ml buffer containing 20 mm Tris pH7.2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM leupeptin, 1 mM DDT, 0.1 mMphenylmethylsulfonyl fluoride using a Potter-Elvehjem Teflon glasstissue homogenizer. Homogenates were sonicated three times for 40 sec,centrifuged at 12,000 g for 15 min, and diluted in sodium dodecylsulfate (SDS) buffer (0.5 M TRIS-HCl pH 6.8, 20% (v/v) glycerol, 4.6%(w/v) SDS). A sample was prepared to contain 350 μg protein and wasapplied to an 8% SDS gel. Proteins were separated by SDS-PAGE andelectroblotting to a nitrocellulose membrane (Pierce, Rockford, Ill.)that was stained by Ponceau solution to ascertain that protein transferto the membrane was complete. The nitrocellulose membranes wereincubated with 3% nonfat dry milk in Tris-buffered saline with 0.1%Tween-20 (TBST) for 1 h, followed by overnight incubation with a mousemonoclonal antibody for bNOS, iNOS, or ecNOS in a 1:400 dilution. Afterrinsing in TBST, membranes were incubated for 1 h with anti-mouse IgGantibody conjugated horseradish peroxidase at a 1:1000 dilution.Membranes were then rinsed with TBST, and bNOS, iNOS, or ecNOS proteinwas detected by diaminobenzidine (DAB) with 0.3% hydrogen peroxide.

Series 3: Immunohistochemical study of ecNOS and bNOS distribution inthe kidney of SHR and WKY. These studies were undertaken to assess thedistribution of ecNOS immunoreactivity in vascular and glomerularcapillary endothelium and bNOS in macula densa cell cytoplasm in SHR andWKY rats. Specifically, differences in constitutive and inducible NOSexpression in various tissues could also indicate the role of NOS inhypertension. After anesthesia, the abdominal aorta of 5 SHR and 5 WKYwas cannulated and the kidneys perfused with 0.154 M NaCl followed byparaformaldehyde lysine periodate (PLP) solution for 5 min, cut intoslices, and immersed into PLP overnight at 4° C. before embedding in wax(polyethylene glycol 400 distearate; Polysciences, Inc., Warrington,Pa.) or paraffin.

Two μm wax sections were processed for light microscopicimmunohistochemistry using the streptavidin-biotin-horseradishperoxidase complex technique (LSAB kit, Dako, Calif.). Briefly, sectionswere dewaxed, rehydrated, and incubated with 3% H₂O₂ for 10 min toeliminate endogenous peroxidase activity. After rinsing in Tris-bufferedsaline with 0.1% Tween 20 (TBST), sections were treated with blockingserum for 10 min and incubated with primary mouse monoclonal antibody ina dilution of 1:100 for bNOS and ecNOS (both from TransductionLaboratories Inc., Lexington, Ky.) for 1 h. After rinsing with TBST, thesections were incubated with the secondary antibody, biotinylated rabbitpolyclonal antibody against mouse immunoglobin (Dako, Denmark), in adilution of 1:600 for 30 min, rinsed, and incubated for 20 min withhorseradish peroxidase (HRP) labeled streptavidin. After rinsing withTBST, HRP was detected by diaminobenzidine (DAB) with hydrogen peroxide.The sections were counter stained with hematoxylin and examined underlight microscopy.

For electron-microscopic (EM) immunocytochemistry using thepost-embedding immunogold procedure, one mm³ blocks of kidney cortex wasdehydrated and embedded in Lowicryl. Ultrathin sections were cut on anultramicrotome, mounted on colloidin-coated nickel grids, and processedfor immunogold labeling. The sections were incubated with 0.1 M NH₄Clfor 1 h, rinsed with buffer solution (0.02 M Tris HCl, 0.15 M NaCl,0.05% Tween 20, adjusted to pH 7.2) for 15 min, and incubated with mousemonoclonal antibody against ecNOS (Transduction Laboratories Inc.,Lexington, Ky.) at a concentration of 1:100 overnight at 4° C. Afterthree 10-min buffer washes, 30 nm gold-labeled goat anti-mouse IgGsecondary antibody (Amersham Life Science, Buckinghamshire, U.K.) wasapplied for 2 h at a dilution of 1:50. Thereafter, the sections werewashed with buffer, incubated with 2% glutaraldehyde/PBS solution for 30min, rinsed with distilled water, counter stained with uranyl acetateand lead citrate, and examined with an electron microscope (Hitachi 7000transmission electron microscope). In order to evaluatesemi-quantitatively the degree of ecNOS immunogold labeling, a blindedobserver assessed EM pictures of sections from 3 SHR and 3 WKY rats. Thenumber of immunogold particles detected overlying epithelial cells werecounted and expressed as the number of particles/μm of glomerularbasement membrane.

Series 4. Effects of inhibition of bNOS on maximal TGF responses in SHRand WKY. These experiments further examined the SHR hypertension modeland whether the enhanced TGF of the SHR kidney is due to a bluntedgeneration of NO by bNOS in the macula densa or whether NOS inhibitionis diminished in the SHR model. Groups of SHR and age-matched WKY ratswere prepared for in vivo micropuncture, microperfusion, and TGF studiesas described in detail previously. In brief, animals were anesthetizedwith thiobarbital (Inactin, 100 mg·kg⁻¹; Research Biochemicals, Inc.,Natick, Mass.). A catheter was placed in a jugular vein for fluidinfusion and in a femoral artery for recording of mean arterial pressure(MAP) from the electrically damped output of a pressure transducer(Statham, Inc.). A tracheotomy tube was inserted and the animals wereallowed to breathe spontaneously. The left kidney was exposed by a flankincision, cleaned of connective tissue, and stabilized in a Lucite cup.This kidney was bathed in 0.154 M NaCl maintained at 37° C. Aftercompletion of surgery, rats were infused with a solution of 0.154 M NaCland 1% albumin at 1.5 ml·h⁻¹ to maintain a euvolemic state.Micropuncture studies were begun after 60 min for stabilization.

For orthograde microperfusion of the loop of Henle (LH), a micropipette(8 μm OD) containing artificial tubular fluid (ATF) stained with FD&Cdye was inserted into a late proximal tubule. Injections of the coloredATF identified the nephron and the direction of flow. An immobile bonewax block was inserted into this micropuncture site via a micropipette(10-15 μm) and connected to a hydraulic drive (Trent Wells, Inc.,LaJolla, Calif.) to halt tubular fluid flow. A perfusion micropipette(6-8 μm) containing ATF and test compounds or vehicle was inserted intothe proximal tubule downstream from the wax block and connected to ananoliter perfusion pump (WPI, Sarasota, Fla.). A pressure micropipette(1-2 μm) was inserted into the proximal tubule upstream from the waxblock to measure proximal stop flow pressure (PSF). Changes in PSF arean index of changes in glomerular capillary hydraulic pressure (P_(GC)).Measurements of PSF were made in each nephron during zero loop perfusionand during perfusion with ATF at 40 nl·min⁻¹, which produces a maximalTGF response, defined as the difference between PSF values recordedduring perfusion of the loop with ATF at 0 and 40 nl·min⁻¹.

The maximal TGF responses were determined in SHR (n=4) and WKY rats(n=4) to perfusion of the LH with ATF+vehicle and contrasted with themaximal TGF responses during perfusion with ATF+7-nitroindazole (7-NI;10⁻⁴ M).

Series 5: Maximal TGF responses during microperfusion of L-arginine inSHR and WKY. L-arginine had previously been shown not to lower bloodpressure or the glomerular filtration rate in the SHR model. However,L-arginine can restore a TGF response to NOS blockade in tested ratsadapted to low salt intake. Therefore, the role of L-arginine was testedin the SHR model. This series examined the effect of a reduced deliveryof L-arginine to the macula densa on NO generation, as assessed inSeries 4. Groups of SHR (n=4) and WKY rats (n=3) were prepared formicroperfusion. PSF was recorded during orthograde LH perfusion at 0 and40 nl·min⁻¹ with ATF+vehicle and ATF+L-arginine (10⁻³ M). (Previousstudies had shown that this was a maximally effective dose.)

Series 6: Effects on maximal TGF responses of microperfusion of Tempolinto the JGA of SHR and WKY. The purpose of this example was todetermine whether oxygen-derived free-radicals in the JGA potentiate TGFin the SHR, and whether this effect can be modulated by the nitroxide,Tempol. Groups of SHR (n=5) and WKY rats (n=5) were prepared for studiesof retrograde microperfusion into the macula densa. As anticipated fromits high membrane permeability, Tempol had rather inconsistent resultswhen perfused orthogradely from the late proximal tubule. Therefore,these studies of TGF were conducted with retrograde microperfusion fromthe early distal (ED) tubule into the macula densa. After identifyingthe nephron with FD&C green, the last proximal convolution was ventedand a wax block placed upstream. A micropipette (8-10 μm OD) wasinserted into the ED tubule upstream from an oil droplet. The loop ofHenle was perfused retrogradely with perfusate entering the macula densasegment directly at 0 and 20 nl·min⁻¹. This represents a maximalactivation for TGF by retrograde perfusion. Preliminary studiesindicated that a dose of Tempol of 10⁻³ M was maximally effective, andthe effects were reversible. Therefore, this dose was used thereafter inthe test animals (Dosage/kg would usually be lower in larger animals).Comparisons were made of maximum TGF responses obtained during perfusionof ATF+vehicle (ethanol) and ATF+Tempol.

Statistical Methods. Values are reported as mean±SEM. An analysis ofvariance (ANOVA) was applied to the within-group data for SHR and WKY;where appropriate, post hoc Dunnett's t tests were applied thereafter.Values were taken as statistically significant at p<0.05.

Results. For Series 1, ecNOS mRNA abundance was consistently greater inouter cortical glomeruli from SHR than WKY, although similar densitieswere apparent for β-actin mRNA. This was confirmed by densitometricanalysis. The cDNA obtained from one glomerulus was analyzed and foundto correspond fully with the published sequence for rat ecNOS.

RT-PCR products corresponding to cDNAs for bNOS were obtained from outercortex of 6 SHR and 6 WKY rat kidneys. The density of the bands obtainedfrom SHR was consistently greater than that for WKY, although similardensities were apparent for β-actin. This difference was confirmed bydensitometric analysis. Analysis of the PCR product from one kidneyconfirmed that it corresponded fully to the published sequence for ratbNOS.

For Series 2, Western analysis of proteins extracted from the outercortex of kidneys of SHR and WKY rats demonstrated bands ofimmunoreactivity corresponding to iNOS and bNOS. A band for ecNOS wasnot consistently detected in the cortex. The expression of bNOS and iNOSimmunoreactive proteins were increased 50-65% in the cortex of the SHRcompared to the WKY.

For Series 3, the distribution of ecNOS and bNOS immunoreactivity in thekidney cortex of SHR and WKY corresponded to previously published datain Sprague-Dawley rats. The ecNOS immunoreactivity was readilydemonstrable in the endothelium of arcuate arteries in the renal cortexof WKY and SHR. In WKY, immunoreactivity was of a relatively modestintensity, whereas in SHR the immunoreactivity in the endotheliumappeared more dense. Immunostaining for ecNOS was also apparent inendothelium of outer cortical arterioles, where it appeared to be lessdense in WKY than in SHR. Using EM immunocytochemistry to assess ecNOSimmunoreactive expression in glomerular capillary endothelium morequantitatively, the number of immunogold particles along the capillarywalls of outer cortical glomeruli was significantly greater in SHR thanWKY (SHR: 0.51±0.05, n=41 vs. WKY: 0.32±0.05, n=40, gold particles·μm⁻¹;p<0.01). Examination of bNOS immunoreactivity showed heavy staining ofthe macula densa cell plaque. There appeared to be less prominent stainin WKY compared to SHR. Kidneys from 5 SHR and 5 WKY rats were testedsystematically for immunocytochemical staining. The results showedclearly stronger macula densa staining for bNOS in SHR compared to WKYin each pair examined by a blinded observer.

The baseline data for the micropuncture/microperfusion studies of ratsof Series 4-6 are shown in Table 1. It is apparent that compared to WKY,SHR rats were of similar body and kidney weight but had consistentlyhigher levels of blood pressure and slightly greater heart rates.Tubuloglomerular feedback (TGF) parameters showed consistently highervalues for proximal stop flow pressure during perfusion of the loop ofHenle at 0 and 40 nl·min⁻¹ and a greater maximal TGF response, asassessed from differences between PSF during perfusion at 0 and 40nl·min⁻¹ in SHR, which averaged 135% of the WKY control.

For Series 4, maximum TGF responses were contrasted in SHR and WKY ratsduring addition of vehicle or 7-NI to orthograde LH perfusates. As shownin Table 2, the maximum TGF responses were greater in SHR than WKYduring perfusion of ATF+vehicle. The addition of 7-NI increased maximalTGF responses consistently in WKY by an average of 39%, but had nosignificant effects on TGF responses of SHR.

For Series 5, TGF responses were contrasted in SHR and WKY duringaddition of L-arginine to orthograde LH perfusates. As shown in Table 3,the maximum TGF responses were greater in SHR compared to WKY duringperfusion of ATF+vehicle. Addition of L-arginine significantly bluntedATF responses of WKY by an average of 18% but had no significant effectson TGF responses of SHR.

For Series 6, TGF responses were contrasted in SHR and WKY duringaddition of the membrane-permeable nitroxide SOD mimetic, Tempol, to LHperfusates. As shown in Table 4, maximum TGF responses were againgreater in SHR than in WKY during retrograde perfusion of ATF+vehicle.Addition of Tempol (10⁻³ M) to the retrograde perfusions of ATF bluntedTGF responses in SHR and WKY rats significantly. However, the bluntingof TGF was significantly (p<0.01) greater in the SHR rats than in WKYrats. When normalized to the initial response, the percentage reductionin TGF with Tempol was again greater in SHR (SHR: −26±2 vs. WKY: −17±3%;p<0.05).

Two additional microperfusion studies were undertaken to assess themechanism of Tempol action in SHR. In the first, local microperfusion ofTempol (10⁻⁴ M) in artificial plasma via the efferent arteriole intoperitubular capillaries was undertaken in 6 rats. A dose of 10⁻⁶ M5-nitroacetylpenacillamine (SNAP), a NO donor compound, wasmicroperfused into the macula densa of the rats. The dose was justsubthreshold, but during Tempol microperfusion the SNAP significantly(p<0.01) blunted TGF response by more than 25%. This demonstratessynergistic interaction between Tempol and a NO donor compound, such asSNAP. Other NO donor compounds include nitroprusside and nitrates.

In the other study, Tempol (10⁻⁴ M artificial plasma) was microperfusedinto efferent arterioles of 7 SHR rats for 4-10 min periods. Whereasbefore Tempol administration there was no significant TGF response tomicroperfusion of the NOS inhibitor 7-nitroindazole (7-NI) into themacula densa at the 10⁻⁴ M concentration, during Tempol microperfusionthere was a robust increase in TGF in the presence of 7-NI of greaterthan 25%. This increase in TGF with 7-NI during Tempol administration issimilar to the increase observed with 7-NI in WKY rats. Therefore,Tempol had normalized the response to NOS blockage in the hypertensivemodel. This also implies that it had normalized the action of NO in thissetting, thereby correcting the defect in the NO action in hypertensionand restoring normal vascular control. This is filly consistent with itsaction on a free radical scavenging agent that thereby protects NO fromradical attack.

In view of the findings from the examples, it is seen that theexpression of both constitutive and inducible NOS isoforms are increasedin the SHR kidney, and that the increase in constitutive NOS isoforms inthe cortex and JGA appears to be transcriptionally regulated since it isaccompanied by an increase in mRNA abundance. Despite this evidence ofenhanced NOS expression in the JGA and/or the renal cortex, the TGFresponses of SHR are exaggerated and are unresponsive either to localblockade of nNOS by microperfusion of 7-NI into macula densa or to localprovision of NOS substrate by microperfusion of L-arginine into themacula densa. These enhanced responses persist after normalization ofthe renal perfusion pressure with a suprarenal aortic clamp andtherefore are not a direct consequence of the elevated BP.

The results with the relatively bNOS-selective antagonist 7-NI, showthat it has no effect on TGF responses of SHR despite potentiating TGFresponses of WKY. Thus, the functional response to NOS inhibition isdiminished in the SHR.

TABLE 1 Whole animal and kidney weights, mean arterial pressure (MAP),heart rate (HR), and tubuloglomerular feedback parameters in WKY and SHRrats used for functional studies Body Kidney MAP PSF (mm Hg) during No.of No. of weight weight (mm HR LH perfusion (nl · min⁻¹) at: Rat strainrats nephrons (g) (g) Hg) (min⁻¹) 0 40 0-40 WKY 10 23 268 ± 8  1.17 ±0.04 116 ± 3 354 ± 6 36.3 ± 0.5 28.0 ± 0.4  8.4 ± 0.3 SHR 13 32 266 ± 151.04 ± 0.06 158 ± 4 378 ± 8 41.0 ± 0.5 29.8 ± 0.4 11.2 ± 0.4 p value nsns <0.001 <0.05 <0.001 <0.01 <0.001 Mean ± SEM values from rats ofseries 4-6. PSF, proximal stop flow pressure.

TABLE 2 Values of proximal stop flow pressure (PSF) as a function ofrate of orthograde perfusion of artificial tubular fluid (ATF) in SHRand WKY: Effects of 7-nitroindazole (7-NI) or renal perfusion pressurePSF (mm Hg) during retrograde LH Rat Added to No. of No. of MAPperfusion (nl · min⁻¹) at: strain ATF rats nephrons (mm Hg) 0 40 0-40WKY Veh 4 8 121 ± 5  37.9 ± 0.8 28.4 ± 0.9  9.5 ± 0.5 7-NI 4 8 38.1 ±0.8 25.0 ± 1.2 13.2 ± 0.7 p value ns <0.05 <0.001 SHR Veh 4 13 168 ± 1141.3 ± 0.9 29.6 ± 0.6 11.8 ± 07 7-NI 4 13 41.0 ± 1.0 29.3 ± 0.7 12.5 ±0.6 Mean ± SEM values from series 4. Veh, vehicle; MAP, mean arterialpressure.

TABLE 3 Values of proximal stop flow pressure (PSF) as a function ofrate of orthograde perfusion of artificial tubular fluid (ATF) in SHRand WKY Effects of L-arginine Added No. of PSF (mm Hg) during LHperfusion Rat to No. of ne- (nl · min⁻¹) at: strain ATF rats phrons 0 400-40 WKY Veh 3 9 36.1 ± 0.7 28.4 ± 0.6  7.7 ± 0.8 L- 3 9 36.1 ± 0.7 29.8± 0.5  6.3 ± 0.4 arginine p value ns ns <0.05 SHR Veh 4 9 41.1 ± 1.230.2 ± 1.1 10.4 ± 0.7 L- 4 9 41.0 ± 1.2 30.4 ± 0.7 10.6 ± 0.7 arginine pvalue ns ns ns Mean ± SEM values. Veh, vehicle.

TABLE 4 Values of proximal stop flow pressure (PSF) as a function ofrate of retrograde perfusion of artificial tubular fluid (ATF) in SHRand WKY: Effects of the nitroxide, superoxide dismutase mimetic, TempolAdded No. of PSF (mm Hg) during LH perfusion Rat to No. of ne- (nl ·min⁻¹) at: strain ATF rats phrons 0 40 0-40 WKY Veh 5 10 34.9 ± 0.8 26.7± 0.7 8.1 ± 0.4 Tempol 5 10 34.7 ± 0.8 28.0 ± 0.9 6.7 ± 0.4 p value nsns <0.05 SHR Veh 5 10 40.3 ± 0.8 28.8 ± 0.6 11.5 ± 0.6  Tempol 5 10 40.8± 0.8 32.1 ± 0.9 8.5 ± 0.8 p value ns <0.05 <0.001 Mean ± SEM values.Veh, vehicle.

Conclusion. Microperfusion of L-arginine into the JGA blunted maximalTGF responses in WKY, yet did not significantly modify responses in SHR.This implies that L-arginine delivery was not limiting for NO generationin the JGA of the SHR. This is consistent with previous findings thatL-arginine does not lower BP or improve the glomerular filtration rate(GFR) of the SHR. The present findings indicate that a deficientdelivery of L-arginine to the JGA cannot explain the enhanced TGF ofouter cortical nephrons of SHR.

Tempol is a low molecular weight, nontoxic compound that equilibratesrapidly between extra- and intracellular compartments, therebyconferring much greater protection against post-ischemic cellular damagethan SOD. Unlike other SOD mimetics, it is not dependent on metals andtherefore is stable in the intracellular environment that contains highMg⁺² concentrations.

Because endothelium-dependent vasodilatation is impaired in the SHR, invitro studies were done in view of the SHR to further evaluate theeffect of Tempol on renal vasoconstriction, vasodilatation andhypertension.

In the first group, the short-term actions of Tempol were determined inanesthetized rats. Baseline mean arterial pressure (MAP) and renalvascular resistance (RVS) were significantly elevated in the SHR (n=6)compared to the WKY (n=6). The following data was obtained:

MAP: SHR=145±4 vs. WKY=118±3 mm Hg,

RVR: SHR=32±4 vs. WKY=10±8 mm Hg/ml/min.

Tempol was administered intravenously at 4 mg/kg and the animals tested

MAP: SHR=108±8 vs. WKY=98±6 mm Hg

RVR: SHR=17±2 vs. WKY=15±1 mm Hg/ml/min

Tempol 12 mg/kg was given intravenously:

MAP: SHR=80±5 vs. WKY=99±7 mm Hg

The longer term effect of administration of Tempol at the rate of 250mg/kg/day given intraperitoneally for 7 days, showed no effect on theMAP in WKY rats, but decreased MAP in the SHR (p<0.01) from 133±2 to120±3 mm Hg.

EXAMPLE 2

The finding that Tempol is an effective treatment in the model ofgenetically-transmitted essential hypertension suggests that Tempol andits derivative forms, as well as other nitroxides, can be used to treatessential hypertension in humans. Tempol and other nitroxides have thespecial potential advantage of not only treating hypertension, but alsocorrecting intra- and extra-cellular oxidative stress simultaneously.

The following compositions are suggestions only and are not meant tolimit the scope of the invention. Oral compositions may contain fillersand, additionally, preservatives along with other inert or activeagents.

A. Compositions for Oral Administration

500 mg Tempol

500 mg starch

5 mg magnesium stearate.

The composition may be placed in capsules which may be enteric coated.Other preparations can include concentrations of Tempol from about 1mg/kg/day to about 500 mg/kg/day. In rats, effective dosagesadministered orally include from about 0.7 to about 15,000 mg/kg/dayorally, or more preferred from about 0.7 to about 1,500 mg/kg/dayorally, or most preferred from about 7 to about 150 mg/kg/day. Inhumans, because of the slower metabolism, the effective dosages ofTempol administered orally include from about 0.07 to about 7,500mg/kg/day orally, or more preferred from about 0.07 to about 750mg/kg/day orally, or most preferred from about 0.7 to about 75mg/kg/day.

B. Compositions for Parenteral Administration

From about 1 gram of Tempol is added to from about 50 to about 100 mlsof 5% dextrose or normal saline or other suitable isotonic solution forintravenous (i.v.) administration. Additional compositions contemplatedfor parenteral use include from about 0.5 mM to about 100 mM Tempol.More preferred would be about 0.5 mM to about 10 mM Tempol administeredin an isotonic vehicle intravenously (i.v.).

Tempol may be administered on a solid support. One example of a solidsupport are patches. Patches for the administration of Tempol can beformulated as adhesive patches containing a nitroxide. For example, thepatch may be a discoid in which a pressure-sensitive silicone adhesivematrix containing the active agent may be covered with a non-permeablebacking. The discoid may either contain the active agent in the adhesiveor may have attached thereto a support made of material such aspolyurethane foam or gauze that will hold the active agent (e.g.,Tempol). Before use, the material containing the active agent would becovered to protect the patch.

C. Compositions for Intravenous Administration

In rats, effective dosages administered intravenously (i.v.) include:(1) from about 0.25 to about 800 mg/kg by i.v. bolus dosing, morepreferred from about 0.25 to about 80 mg/kg by i.v. bolus dosing, andmost preferred from about 2.5 mg/kg to about 8 mg/kg by i.v. bolusdosing; and (2) from about 0.5 to about 2,000 mg/kg/hr by i.v. infusion,more preferred from about 0.5 to about 200 mg/kg/hour by i.v. infusion,and most preferred from about 5 to about 20 mg/kg/hour by i.v. infusion.In humans, because of the slower metabolism, the effective dosages ofTempol administered intravenously include: (1) from about 0.025 to about400 mg/kg by i.v. bolus dosing, more preferred from about 0.025 to about40 mg/kg by i.v. bolus dosing, and most preferred from about 0.25 mg/kgto about 4 mg/kg by i.v. bolus dosing; and (2) from about 0.05 to about1,000 mg/kg/hr by i.v. infusion, more preferred from about 0.05 to about100 mg/kg/hour by i.v. infusion, and most preferred from about 0.5 toabout 10 mg/kg/hour by i.v. infusion.

D. Compositions for Dermal Administration

A patch or other solid support composed of trilaminate of an adhesivematrix sandwiched between a non-permeable backing and a protectivecovering layer is prepared in the following manner:

Two grams of Tempol is applied to from about 5 grams of apressure-sensitive silicone adhesive composition BIOPSA™ Q7-2920 (DowCorning Corp., Midland, Mich., U.S.A.). The adhesive is applied to apolyester film to provide in successive layers to provide about 200 mgof active agent per cm². The film containing the adhesive is then madeinto a patch of 10 cm². The patch is covered with a protective layer tobe removed before application of the patch.

Patches may be prepared containing permeation enhancers such ascyclodextrin, butylated hydroxyanisole, or butylated hydroxytoluene.However, it should be remembered that the active agents of thisinvention are effective on application to the epidermal tissue. When thepatches are to be applied to thin or abraded skin, there is little needto add a permeation enhancer.

EXAMPLE 3

Materials and Methods. In Examples 3 to 6, groups of male SHR and WKYrats (200 to 300 g) were maintained on tap water and standard chow(Harlan-Teklad Inc.). In Example 3, renal hemodynamics and MAP duringbolus intravenous injection of Tempol were compared in anesthetized SHRand WKY. In order to do this experiment, WKY (n=6) and SHR (n=6) wereanesthetized with thiobutabarbital (100 mg/kg i.p., Inactin, ResearchBiochemicals International) and maintained at 37° C. on aservocontrolled heated rodent operating table. A tracheostomy wasperformed with polyethylene PE-240 tubing, and the left jugular vein andcarotid artery were cannulated with PE-50 tubing. Intravenous infusionof 1% albumin dissolved in 0.154 M NaCl solution was infused at 2 mL/hi.v. to maintain an euvolemic state. A midline incision was made, andthe left renal artery was isolated. A blood-flow probe was placed aroundthe renal artery and connected to a transit-time blood flowmeter (1RB,Transonic Systems Inc.). We have previously shown that this method ofmeasuring real-time changes in RBF is valid in the rat (Welch et al.,1995 Am. J. Physiol. 37: F175-F178).

MAP was continuously recorded from the carotid artery using a Stathampressure transducer (model P23, Gould Instruments) and MACLab dataacquisition program. After 60 minutes of equilibration, there was abasal period for measurement of MAP and RBF over 30 minutes. Then theMAP and RBF responses to Tempol at 24 and 72 μmol/kg i.v. weredetermined.

Statistics. All values shown are mean±SE. ANOVA was used to determinestatistical significance in groups 1 and 2. Student's t test was used todetermine significance in groups 3 and 4, where the comparison waslimited to two observatims. P<0.05 was considered statisticallysignificant. The statistical methods used are the same for Examples 3 to6.

Results. FIG. 1 shows the MAP during baseline conditions and afterintravenous injections of Tempol at 24 and 72 μmol/kg in WKY and SHR.Baseline MAP was significantly elevated in SHR compared with WKY (145±4versus 118±3 mm Hg, respectively; P<0.05). Low-dose Tempol (24 μmol/kgIV) had no effect in either the WKY (114±5 mm Hg) or SHR (147±4 mm Hg).However, higher-dose Tempol normalized the MAP of the SHR to the levelof WKY. Tempol (72 μmol/kg IV) significantly (P<0.05) decreased MAP by11% in WKY (96±6 mm Hg) and by 28% in SHR (104±9 mm Hg).

Renal hemodynamics were studied during basal conditions and infusion ofTempol at 24 and 72 μmol/kg in WKY and SHR. Baseline RBF was similarbetween groups (WKY, 7.1±0.7; SHR, 6.8±1.0 mL/min) and was not affectedduring Tempol (WKY, 6.6±0.7; SHR, 6.7±0.8 mL/min). In contrast, baselineRVR was significantly increased in SHR compared with WKY (24±3 versus17±1 mm Hg·mL⁻¹·min⁻¹, respectively; P<0.05). Low-dose Tempol had noeffect on RVR in either group (WKY, 17±1; SHR, 24±3 mm Hg·mL⁻¹·min⁻¹).However, higher-dose Tempol normalized the RVR of the SHR to the levelof WKY. Tempol at 72 μmol/kg significantly (P<0.05) decreased RVR by 29%in SHR (17±2 mm Hg·mL⁻¹·min⁻¹), while having a minimal effect in WKY(15±1 mm Hg·mL⁻¹·min⁻¹).

Previous studies investigating the short-term actions of O₂ ⁻ on bloodpressure in SHR demonstrated that bolus injection of a xanthine oxidaseinhibitor to block the formation of O₂ ⁻ from xanthine or CuZn SODacutely decreased MAP in the SHR; however, results for WKY were notreported (Miyamoto et al., 1996; Nakazono et al, 1991 Proc. Nat'l Acad.Sci. USA 88: 10045-10048). This treatment corrects O₂ ⁻ generation onlyfrom xanthine oxidase, whereas the proposed mechanism of action ofTempol as a SOD mimetic predicts that it should correct O₂ ⁻overproduction from all sources. Moreover, it can gain access to bothintra- and extra-cellular sites, and will therefore correct oxidativestress arising intra- or extra-cellularly. Most other antioxidants, suchas vitamin C and SOD act purely extracellularly. Therefore, we comparedthe effect of scavenging O₂ ⁻ on MAP in SHR to their genetic controlWKY. We show that acute Tempol administration normalized MAP and RVR inSHR to the level of WKY.

Earlier studies have established a role for O₂ ⁻ in the aorta andmesenteric arterioles of SHR (Auch-Schwelk et al., 1989 Hypertens. 13:859-864; Miyamoto et al., 1996; Grunfeld et al., 1995; and Susuki etal., 1995 Hypertens. 25: 1083-1089). However, the kidneys play animportant role in the development and maintenance of hypertension.Tempol vasodilated the renal vasculature in SHR more than in WKY. Undercontrol conditions, RVR was significantly elevated in SHR, and Tempolnormalized RVR in SHR to the level of WKY. Because Tempol reduced MAPwithout changing RBF, renal vasodilation was inferred. The RVR responseto Tempol may be a result of RBF autoregulation. Whether Tempol directlyor indirectly decreases RVR in SHR remains to be further elucidated.This is the first demonstration that the elevated renal vascularresistance (RVR) of a model of hypertension (e.g., SHR) can be correctedby a therapy directed at scavenging of intra-cellular and extra-cellularO₂ ⁻.

EXAMPLE 4

Materials and Methods. In Example 4, the MAP during constant intravenousinfusion of Tempol was compared in anesthetized SHR and WKY. Todetermine the dose-response relationship for Tempol, MAP was measuredduring basal conditions and during intravenous infusion of Tempol at1.8, 18, 180, and 1800 μmol·kg⁻¹·h⁻¹ for 30 minutes in anesthetized WKY(n=6) and SHR (n=6).

FIG. 2 illustrates the dose-response relationship between Tempol at 1.8,18, 180, 1,800 μmol·kg⁻¹·h⁻¹ and MAP in WKY and SHR. Baseline MAP wasagain significantly (P<0.05) elevated in the SHR (166±7 mm Hg) comparedwith WKY (121±4 mm Hg). Tempol dose-dependently decreased MAP in WKY andSHR, with SHR having a greater sensitivity and responsiveness to Tempolinfusion. The highest dose of Tempol (1,800 μmol·kg⁻¹·h⁻¹) normalizedthe MAP of SHR (72±10 mm Hg) to the level of WKY (71±3 mm Hg).

EXAMPLE 5

Materials and Methods. In Example 5, the role of NO in the MAP responseto constant Tempol infusion in SHR was investigated To determine whetherO₂ ⁻ increases MAP through interaction with the NO pathway, the MAPresponse to Tempol was determined in anesthetized SHR (n=6) and in SHRpretreated with the NO synthase inhibitor L-NAME (11 μmol·kg⁻¹·min⁻¹,n=5). To ensure that any change in the MAP response to Tempol in SHRduring L-NAME administration was not due solely to an increase in MAPand vascular tone, the protocol was repeated in SHR infused withnorepinephrine (31 μmol·kg⁻¹·min⁻¹, n=6). In all rats, MAP was measuredduring basal conditions; during 20 minutes of pretreatment with eithersaline vehicle, L-NAME, or norepinephrine; and after 30 minutes ofconstant Tempol infusion (180 μmol·kg⁻¹·h⁻¹).

Results. The percent change in MAP was compared in SHR pretreated withisotonic saline vehicle (2 mL/h i.v.) or the NO synthesis inhibitorL-NAME (11 μmol·kg⁻¹·h⁻¹ i.v.). As in the previous group, infusion ofTempol (180 μmol·kg⁻¹·min⁻¹) for 30 minutes significantly decreased MAPby 32% in SHR (121±17 mm Hg, P<0.05). In marked contrast, the NOsynthesis inhibitor L-NAME abolished the MAP response to Tempol. Twentyminutes of L-NAME infusion alone increased MAP by 18% from 158±11 to187±8 mm Hg, and MAP remained unchanged during Tempol infusion (186±4 mmHg). Time control studies in a separate group of SHR showed that MAPremained steady during L-NAME infusion (change in MAP at 50 min,0.3±3.3%; NS).

To investigate whether the failure of Tempol to lower MAP inL-NAME-infused rats was a consequence of the severe vasoconstriction andhypertension, the protocol was repeated in SHR infused withnorepinephrine (31 nmol·kg⁻¹·min⁻¹) in place of L-NAME. Norepinephrineincreased MAP by 15% from 164±4 to 188±7 mm Hg. This was similar to theincrease with L-NAME. However, Tempol significantly decreased MAP by 14%(161±7 mm Hg, P<0.05) in SHR infused with norepinephrine. Time-controlstudies, in a separate group of SHR, showed that MAP remained steadyduring norepinephrine infusion (change in MAP at 50 min, 2.0±0.0%; NS).

Therefore, this example shows that the antihypertensive response mustdepend on NOS, because it was blocked by NO synthesis inhibition. Theintravenous infusion of Tempol decreases MAP by 32% in SHR, and thisresponse is blocked in SHR rats pretreated with the NO synthaseinhibitor L-NAME. It has also been shown that the negative response toTempol during L-NAME was not merely due to an increase in systemicvascular resistance and blood pressure, because of the MAP response toTEMPOL in SHR infused with norepinephrine. In SHR pretreated withnorepinephrine, which produced a similar increase in MAP, Tempol reducedMAP by 14%. Previous investigators have shown that catecholamines,including norepinephrine, have antioxidant properties. Becausenorepinephrine is an antioxidant, the addition of another antioxidantwould not have as marked an effect as Tempol administered alone to rats.For this reason, Tempol may have been less effective in lowering MAP inSHR pretreated with norepinephrine (14%) than in normal SHR (32%).Overall, these data suggest that NO plays an important role in mediatingthe antihypertensive actions of scavenging of O₂ ⁻.

There are several possible mechanisms that should be considered by whichNO mediates the antihypertensive actions of Tempol. First, could Tempoldirectly donate NO? This possible mechanism has been proven incorrect,because Tempol does not decompose to NO (Landino et al., 1996 Proc. NatlAcad. Sci. USA 93: 15069-15074). Second, scavenging of O₂ ⁻ increasesthe half-life of NO. Gryglewski et al. (1986 Nature 320: 454-456) showedthat O₂ ⁻ is important in the breakdown of NO to peroxynitrite, andRubanyi et al. (1986) demonstrated that O₂ ⁻ inactivates NO in coronaryartery rings. There are several possible sources of O₂ ⁻, includingxanthine oxidase, NADPH oxidase, incomplete electron transport and evenbrain NOS (Samuni et al., 1991 Clin. Invest. 1526-1530). The source ofO₂ ⁻ in this study remains unclear. However, because previous studiessuggest a role of O₂ ⁻ released from the vasculature in SHR, brain NOSdoes not appear to be the major source of O₂ ⁻. As a result of thepowerful interaction between O₂ ⁻ and NO, Tempol may prolong thehalf-life of NO and thus allow it to exert a more powerful vasodilatoryaction. Finally, by blocking the formation of peroxynitrite, Tempol mayinhibit the production of vasoconstrictor endoperoxides that arestimulated by peroxynitrite in macrophages (Landino et al., 1996).Nevertheless, this is the first study to show that scavenging of O₂ ⁻both extracellularly and intracellularly with a membrane permeable SODmimetic, such as Tempol, normalizes the RVR and MAP of SHR. Other SODnitroxide mimetics are also considered (see Schnackenberg et al., 1998).

EXAMPLE 7

Materials and Methods. Groups of male SHR and WKY rats (250±10 g) weremaintained on tap water and a standard chow (Ralston-Purina Co., sodiumcontent 0.3 g/100 g). Rats were divided into four groups: WKY givenvehicle (n=7), SHR given vehicle (n=8), WKY given Tempol (n=6), and SHRgiven Tempol (n=8). Tempol is readily soluble in water and wasadministered in the drinking water (1 mM) for two weeks. After eithercontrol or Tempol administration, rats were maintained in metaboliccages for 24 hours. Urine was collected in containers with 10 μl of 2 mMethylenediaminetetraacetic acid (EDTA) to prevent ex vivo production of8-iso-prostaglandin F_(2α) (8-ISO). Urine was centrifuged at 1,000 rpmfor 10 min at 4° C. and stored in aliquots at −80° C. until assayed.

Thereafter, WKY and SHR were anesthetized with thiobutabarbital (100mg/kg, i.p., Inactin, Research Biochemicals International) andmaintained at 37° C. on a servo-controlled heated rodent operatingtable. A tracheostomy was performed with polyethylene PE-240 tubing andthe left jugular vein and carotid artery were cannulated with PE-50tubing. A 1% albumin solution in 0.154 M NaCl was infused at 2 ml/h,i.v. to maintain a euvolemic state. A midline incision was made and theleft renal artery was isolated. A blood flow probe was placed around therenal artery and connected to a transit-time blood flowmeter (IRB,Transonic Systems, Inc.). We have previously shown that this method ofmeasuring real-time changes in renal blood flow (RBF) is valid in therat. Mean arterial pressure (MAP) and heart rate (HR) were continuouslyrecorded from the carotid artery using a Statham pressure transducer(model P23, Gould Instruments) and MACLab data acquisition software.Glomerular filtration rate (GFR) was determined from the clearance of[³H]-inulin. Following surgery and a 60 min equilibration period, MAP,HR, GFR, and RBF were measured over 30 minutes and the data wasaveraged.

Statistics. All values shown are mean±SE. ANOVA was used to determinestatistical significance in groups 1 and 2. Student's t test was used todetermine significance in groups 3 and 4, where the comparison waslimited to two observatims. P<0.05 was considered statisticallysignificant.

Results. Rats maintained on Tempol given in the drinking water for 2weeks had similar dietary consumption as control rats drinking wateralone. There was no significant difference between food intake (Control:25±1 vs. Tempol: 24±1 g/day) or body weight gain (Control: 69±4 vs.Tempol: 66±3 g/14 days), but water intake was increased modestly in theTempol treated groups (Control: 34±2 vs. Tempol: 43±3 ml/day, p<0.05).Water intake was increased similarly in Tempol-treated WKY and SHR.

Mean arterial pressure in WKY and SHR is represented in FIG. 4. Undernormal conditions, MAP in SHR was increased by 41% compared to WKY (SHR:162±8 vs. WKY: 115±5 mm Hg, p<0.001). After two weeks of Tempoladministration, MAP was reduced in SHR to a value that was notsignificantly different from WKY (SHR: 134±6 vs. WKY: 118±7 mm Hg). MAPin SHR given Tempol was significantly lower by 18% compared to normalSHR. Analysis of variance showed that Tempol specifically andsignificantly (p<0.05) decreased MAP in SHR. Heart rate wassignificantly (p<0.001) elevated in SHR (420±6 bts/min) compared to WKY(374±9 bts/min) during control conditions and was not changed by Tempol(SHR: 414±9 vs. WKY: 373±8 bts/min). Two weeks of Tempol administrationin the drinking water (1 mM) to SHR (n=8) decreased MAP by 18% (162±8 to134±6 mm Hg, p<0.05), increased GFR by 17% (1.6±0.2 to 1.9±0.3 ml/min)and decreased UV8-ISO by 39% (9.8±0.7 to 6.0±0.7 ng/24 hr, p<0.05).

TABLE 5 RBF GFR RVR UV U_(Na)V Group ml/min ml/min mm Hg/ml/min ml/24 hrmmol/24 hr Control WKY 8.8 ± 0.7  3.0 ± 0.4 13.5 ± 1.0  14.2 ± 1.5 2.6 ±0.3 Control SHR 5.8 ± 0.6*  1.6 ± 0.2* 29.4 ± 2.7* 13.8 ± 2.2 2.0 ± 0.3Tempol WKY 8.8 ± 0.9  2.5 ± 0.4 14.2 ± 1.4  16.7 ± 2.3 2.4 ± 0.6 TempolSHR 5.6 ± 0.5* 2.0 ± 0.2 26.4 ± 2.6* 15.2 ± 1.7 2.1 ± 0.2

Table 5 depicts renal hemodynamic and excretory function during normalconditions and after 2 weeks of Tempol administration in the drinkingwater. Under normal conditions, the RBF of SHR was decreased by 34%(SHR: 5.8±0.6 vs. WKY: 8.8±0.7 ml/min, p<0.01), the GFR was decreased by47% (SHR: 1.6±0.2 vs. WKY: 3.0±0.4 ml/min, p<0.05), and the RVR wasincreased by 117% (SHR: 29.4±2.7 vs. WKY: 13.5±1.0 mm Hg/ml/min,p<0.001). After two weeks of Tempol administration, there were nosignificant changes in renal hemodynamics in SHR, although there weretendencies towards a decrease in RVR (16%) and an increase in GFR (17%),such that there was no longer a significant difference in GFR betweenSHR and WKY. Tempol had no marked effects on renal hemodynamics in WKY.Renal excretory function was not significantly different between WKY andSHR during control conditions or Tempol administration.

This example demonstrates that prolonged oral Tempol therapy selectivelylowers the BP in a rat model of essential hypertension, but withouteffect on the WKY control rats. Importantly, the marker of O₂ ⁻generation (e.g., excretion of 8-ISO) was increased in the SHR model,yet Tempol selectively reduced this marker to the value observed in theWKY control rats over the two week period of its administration. This isthe first demonstration that steady-state administration of an agent(e.g., Tempol) can simultaneously and selectively correct hypertensionand oxidative stress in a model of human essential hypertension. Itprovides a strong, rational basis for these forms of therapy in humanessential hypertension.

Increased ROS was detected in our study of the rat model ofmild/moderate essential hypertension (e.g., the SHR rat). It is likelythat the ROS is more severe and ROS reversal is more important andurgent in the more severe forms of hypertension, such as accelerated,drug-resistant or malignant hypertension, or hypertensive urgencies,emergencies and crises. Therefore, the more severe forms ofhypertension, that are often accompanied by dysfunction of the heart orbrain, may be the forms of hypertension best treated by Tempolcontaining compositions.

In the above setting, patients often require intravenous therapy in ahospital under close monitoring in an intensive care unit. The drug ofchoice currently is usually the NO donor compound sodium nitroprusside(Nipride). Our data demonstrates that Tempol acts synergistically withS-nitrosopenacillamine (SNAP) in our rat studies to reduce glomerularcapillary pressure by relaxing the efferent arteriole of the kidney.This provides a potential basis for combining Tempol with NO providingagents, such as Nipride, in certain patients.

Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims. Allcited patents and publications referred to in this application areherein incorporated by reference in their entirety. This applicationalso incorporates in its entirety patent application Ser. No. 08/933,379filed Sep. 19, 1997.

I claim:
 1. A method of treating hypertension, a hypertensive emergency,a hypertensive crisis or essential hypertension in a patient comprisingthe step of administering to said patient a blood pressure loweringeffective amount of a nitroxide in a pharmaceutically acceptablecarrier, wherein said nitroxide is selected from the group consisting ofTEMPO, DOXYL and PROXYL.
 2. The method of claim 1, wherein saidnitroxide in a pharmaceutically acceptable carrier is administeredorally.
 3. The method of claim 1, wherein said nitroxide in apharmaceutically acceptable carrier is administered parenterally.
 4. Themethod of claim 1, wherein said nitroxide in a pharmaceuticallyacceptable carrier is administered intravenously.
 5. The method of claim1, wherein said nitroxide in a pharmaceutically acceptable carrier isadministered transdermally.
 6. A method of treating hypertension, ahypertensive emergency, a hypertensive crisis or essential hypertensioncomprising administering to a patient in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising Tempol,wherein said Tempol is formulated to deliver a bolus dose intravenouslyof about 0.025 mg/kg to about 400 mg/kg.
 7. A method of treatinghypertension, a hypertensive emergency, a hypertensive crisis oressential hypertension comprising administering to a patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising Tempol, wherein said Tempol is formulated todeliver a bolus dose intravenously of about 0.025 mg/kg to about 40mg/kg.
 8. A method of treating hypertension, a hypertensive emergency, ahypertensive crisis or essential hypertension comprising administeringto a patient in need thereof a therapeutically effective amount of apharmaceutical composition comprising Tempol, wherein said Tempol isformulated to deliver a bolus dose intravenously of about 0.25 mg/kg toabout 4 mg/kg.
 9. A method of treating hypertension, a hypertensiveemergency, a hypertensive crisis or essential hypertension comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising Tempol, wherein saidTempol is formulated to deliver an intravenous dose of about 0.05mg/kg/hr to about 1000 mg/kg/hr by intravenous infusion.
 10. A method oftreating hypertension, a hypertensive emergency, a hypertensive crisisor essential hypertension comprising administering to a patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising Tempol, wherein said Tempol is formulated todeliver an intravenous dose of about 0.05 mg/kg/hr to about 100 mg/kg/hrby intravenous infusion.
 11. A method of treating hypertension, ahypertensive emergency, a hypertensive crisis or essential hypertensioncomprising administering to a patient in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising Tempol,wherein said Tempol is formulated to deliver an intravenous dose ofabout 0.5 mg/kg/hr to about 10 mg/kg/hr by intravenous infusion.
 12. Amethod of treating essential hypertension comprising administering to apatient in need thereof a therapeutically effective amount of a patchfor dermal administration of Tempol wherein said Tempol is formulated toprovide about 200 mg per square centimeter of said patch.
 13. A methodof treating hypertension, a hypertensive emergency, a hypertensivecrisis or essential hypertension comprising the step of administering toa subject in need thereof an effective amount of a pharmaceuticalcomposition comprising Tempo, Tempol, Doxyl or Proxyl, wherein saidTempo, Tempol, Doxyl or Proxyl is formulated to deliver an oral dailydose of about 0.07 mg/kg/day to about 7500 mg/kg/day.
 14. The method ofclaim 13, wherein said Tempo, Tempol, Doxyl or Proxyl is formulated todeliver an oral daily dose of about 0.07 mg/kg/day to about 750mg/kg/day.
 15. The method of claim 14, wherein said Tempo, Tempol, Doxylor Proxyl is formulated to deliver an oral daily dose of about 0.7mg/kg/day to about 75 mg/kg/day.
 16. A method of treating hypertension,a hypertensive emergency, a hypertensive crisis or essentialhypertension comprising the step of administering to a subject in needthereof an effective amount of a pharmaceutical composition comprisingTempo, Tempol, Doxyl or Proxyl, wherein said Tempo, Tempol, Doxyl orProxyl is formulated to deliver an intravenous bolus dose of about 0.025mg/kg to about 400 mg/kg.
 17. The method of claim 16, wherein saidTempo, Tempol, Doxyl or Proxyl is formulated to deliver an intravenousbolus dose of about 0.025 mg/kg to about 40 mg/kg.
 18. The method ofclaim 17, wherein said Tempo, Tempol, Doxyl or Proxyl is formulated todeliver an intravenous bolus dose of about 0.25 mg/kg to about 4 mg/kg.19. A method of treating hypertension, a hypertensive emergency, ahypertensive crisis or essential hypertension comprising the step ofadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising Tempo, Tempol, Doxyl or Proxyl,wherein said Tempo, Tempol, Doxyl or Proxyl is formulated to deliver adose of about 0.05 mg/kg/hr to about 1000 mg/kg/hr by intravenousinfusion.
 20. The method of claim 19, wherein said Tempo, Tempol, Doxylor Proxyl is formulated to deliver a dose of about 0.05 mg/kg/hr toabout 100 mg/kg/hr by intravenous infusion.
 21. The method of claim 20,wherein said Tempo, Tempol, Doxyl or Proxyl is formulated to deliver adose of about 0.5 mg/kg/hr to about 10 mg/kg/hr by intravenous infusion.22. A method of treating hypertension, a hypertensive emergency, ahypertensive crisis or essential hypertension comprising the step ofadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising Tempo, Tempol, Doxyl or Proxyl,wherein said Tempo, Tempol, Doxyl or Proxyl is formulated for deliverytransdermally via a patch, wherein said Tempo, Tempol, Doxyl or Proxylis present on said patch at a concentration of about 200 mg per squarecentimeter of said patch.